Biomedical subjects

Weirong Xing

Publications and source records attributed to Weirong Xing.

8 recordsLinked to original sources

PubMed2006

A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro.

We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific gene osterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/ Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency, transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an increase in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.

3T3 Cells↗

PubMed2005

Global gene expression analysis in the bones reveals involvement of several novel genes and pathways in mediating an anabolic response of mechanical loading in mice.

To identify the genes and signal pathways responsible for mechanical loading-induced bone formation, we evaluated differential gene expression on a global basis in the tibias of C57BL/6J (B6) mice after four days of four-point bending. We applied mechanical loads to the right tibias of the B6 mice at 9 N, 2 Hz for 36 cycles per day, with the left tibias used as unloaded controls. RNA from the tibias was harvested 24 h after last stimulation and subjected to microarray. Of the 20,280 transcripts hybridized to the array, 346 were differentially expressed in the loaded bones compared to the controls. The validity of the microarray data was established with the increased expression of bone-related genes such as pleiotrophin, osteoglycin, and legumain upon four-point bending and confirmation of increased expression of selected genes by real-time PCR. The list of differentially expressed genes includes genes involved in cell growth, differentiation, adhesion, proteolysis, as well as signaling molecules of receptors for growth factors, integrin, Ephrin B2, endothelin, and adhesion G protein coupled receptor. Pathway analyses suggested that 28 out of the 346 genes exhibited a direct biological association. Among the biological network, fibronectin and pleitrophin function as important signaling molecules in regulating periosteal bone formation and resorption in response to four-point bending. Furthermore, some expressed sequence tags (ESTs) with no prior known function have been identified as potential mediators of mechanotransduction signaling pathways. Further studies on these previously unknown genes will improve our understanding of the molecular pathways and mechanisms involved in bone's response to mechanical stress.

Anabolic Agents↗

PubMed2004

HSV-1 amplicon-mediated transfer of 128-kb BMP-2 genomic locus stimulates osteoblast differentiation in vitro.

In previous studies, we developed mouse genetic models and discovered genetic components of quantitative trait loci on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and fracture healing. However, these regions contain dozens of genes in several overlapping bacterial artificial chromosomes (BACs) and are difficult to clone by physical cloning strategies. A feasible and efficient approach of identifying candidate genes is to transfer the genomic loci in BAC clones into mammalian cells for functional studies. In this study, we retrofitted a BAC construct into herpes simplex virus-1 amplicon and packaged it into an infectious BAC (iBAC) to test gene function in a cell-based system, using a 128-kb clone containing the complete bone morphogenetic protein-2 (BMP-2) gene. We transduced MC3T3-E1 cells with the iBAC bearing BMP-2 gene and examined transgene expression and function. Our results have demonstrated that an iBAC can efficiently deliver a BMP-2 genomic locus into preosteoblast cells and express functional BMP-2 protein, inducing a phenotype of cell differentiation, as indicated by an increase in alkaline phosphatase activity. Therefore, this experimental system provides a rapid, efficient cell-based model of high-throughput phenotypic screening to identify the BAC clones from physically mapped regions that are important for osteoblast differentiation. It also illustrates the potential of iBAC technology in functional testing of single nucleotide polymorphisms located in the distal promoter or/and intron regions responsible for low bone density.

3T3 Cells↗

PubMed2003

Role of follitropin receptor signaling in nuclear protein transitions and chromatin condensation during spermatogenesis.

Follitropin receptor (FSHR) in testicular Sertoli cells mediates signaling by pituitary follitropin (FSH) promoting intercellular communication with germ cells for normal spermatogenesis. Using receptor knockout mice we examined changes in sperm nucleoproteins and chromatin architecture. The expressions of transition proteins 1/2 (TP1/2) and protamine-2 (PRM-2) were greatly diminished at 21 days, but returned to normal at 35 days and 3 months after birth. However, protein components in chromatin were quite different. Western blots detected a reduction in PRM1/2 and prolonged retention of mono-ubiquitinated histone 2A (uH2A) in the epididymal sperm from adult mutants. Two forms of mono- and poly-uH2A were present in sonication-resistant testicular spermatids in normal mice, whereas only an elevated mono-uH2A was detectable in mutants. Decrease in PRM1/2 and retention of mono-uH2A was coincident with reduction in TP1/2 in premature spermatids. Thus lack of FSHR signaling impairs expression of TP1/2 and PRM-2 at an early stage of post-natal development causing delayed spermatogenesis. In the adult, absence of FSHR signaling prolongs retention of mono-uH2A, leading to impair transition of basic nucleoproteins and chromatin remodeling during mouse spermatogenesis.

Animals↗

PubMed2002

Cross talk of two Krupple transcription factors regulates expression of the ovine FSH receptor gene.

The follicle-stimulating hormone receptor (FSHR) in gonadal cells is required for normal folliculogenesis and spermatogenesis. To understand its regulation, we identified a CACC-box from -46 to -67 of the ovine FSHR promoter. Antibody supershift with a 22-bp DNA probe and nuclear extract from a Sertoli cell line demonstrated that a testis-specific zinc finger protein, ZNF202, might be one of the binding proteins. Western blots using ZNF202 antibody and Southwestern blot analyses with the DNA probe detected the same 60kDa protein in both Sertoli and ovarian granulosa cell lines. Gel shift assays also revealed that the DNA-protein complex from Sertoli cells overexpressing the human Ras-responsive element binding protein-1 (RREB-1) migrated the same way as the complex containing endogenous CACC-box binding protein. Transfection studies indicated that ZNF202 repressed ovine FSHR promoter activity whereas RREB-1 was likely to function as an activator. These data suggest that selective expression and cross talk of functionally distinctive Krupple transcription factors could regulate tissue- and stage-specific expression of FSHR gene.

Animals↗

PubMed2002

Orphan receptor chicken ovalbumin upstream promoter transcription factors inhibit steroid factor-1, upstream stimulatory factor, and activator protein-1 activation of ovine follicle-stimulating hormone receptor expression via composite cis-elements.

The FSH receptor (FSHR) is selectively expressed in the granulosa and Sertoli cells in a development-dependent manner. Little is known regarding how the regulatory factors balance expression of this gene in ovarian cycles or spermatogenic stages. We have used the ovine FSHR promoter as a model system and identified a third regulatory element (RE-3) located at -197 to -171 of the strongest promoter. Gel mobility shift and antibody supershift assays demonstrated that nuclear factors c-Fos/c-Jun, steroidogenic factor-1 (SF-1), upstream stimulatory factor-1/2 (USF-1/2), and chicken ovalbumin upstream promoter transcription factor-1/2 (COUP-TFI/II) potentially bound to RE-3. We have also extended our previous observations by showing that a sequence containing an E-box was not only bound by USF proteins but also recognized by COUP-TF orphan receptors. Functional studies demonstrated that USF-1/2, c-Fos/c-Jun, and SF-1 were activators, whereas COUP-TFs were repressors. Our studies indicated that RE-3 mediated SF-1 activation as well as phorbol 12-myristate 13-acetate stimulation, whereas COUP-TFs inhibited AP-1, USFs, and SF-1 activation. We also demonstrated that both COUP-TF-binding sites in the core promoter were required for the bipartite elements to oppose their competitor binding. These data suggest a mechanism by which positive and negative regulators compete for the common regulatory elements, providing antagonistic pathways that might govern the expression of FSHR in gonadal cells.

Animals↗

PubMed2002

Retinoic acid mediates transcriptional repression of ovine follicle-stimulating hormone receptor gene via a pleiotropic nuclear receptor response element.

The FSH receptor (FSHR) and retinoid receptors are critical regulators of gonadal function. Unlike the latter, the FSH receptors are expressed exclusively in ovarian granulosa and testicular Sertoli cells in a developmental fashion. Toward understanding the nature of various transcription factors that direct a tissue- and stage-specific expression of the FSHR gene, we have studied FP4, one of the two footprinting regions (FP3 and FP4) mapped at -241 to -269 and -284 to -303, respectively, upstream of the transcription start site of the ovine FSHR gene. Gel mobility shift assays with FP4 probe revealed two sequence-specific DNA-protein complexes in the presence of nuclear extracts from two immortal gonadal cell lines. Antibody supershift assays demonstrated that retinoic acid receptor (RAR) was involved in the complex 1 whereas steroidogenic factor-1 (SF-1) was present in the complex 2. Mutation studies revealed that DNA binding sites for RAR and SF-1 were overlapping each other within a 19-base pair length of nucleotide sequence of FP4, and a mutation in the half RAR binding site seriously affected SF-1 binding. Reporter assays showed that FP4 conferred SF-1 transactivation as well as RAR-mediated, ligand-dependent repression. Overexpression of SF-1 in a transformed Sertoli cell line partially overcame RAR-mediated suppression. For the first time, our studies reveal a direct retinoid modulation of the gonadotropin receptor promoter and suggest a mechanism by which activators and repressors compete for composite elements providing antagonistic pathways that could modulate the expression of FSHR.

Animals↗

PubMed2002

Endocrine alterations and signaling changes associated with declining ovarian function and advanced biological aging in follicle-stimulating hormone receptor haploinsufficient mice.

Reproductive aging in female mammals is characterized by a progressive decline in fertility due to loss of follicles and reduced ovarian steroidogenesis. In this study we examined some of the endocrine and signaling parameters that might contribute to a decrease in ovulation and reproductive performance of mice with haploinsufficiency of the FSH receptor (FSH-R). For this purpose we compared ovarian changes and hormone levels in FSH-R heterozygous (+/-) and wild-type mice of different ages (3, 7, and 12 mo). Hormone-induced ovulations in immature and 3-mo-old +/- mice were consistently lower. The number of corpora lutea (CL) were lower at 3 and 7 mo, and none were present in 1-yr-old +/- females. The plasma steroid and gonadotropin levels exhibited changes associated with typical ovarian aging. Plasma FSH and LH levels were higher in 7-mo-old +/- mice, but FSH levels continued to rise in both genotypes by 1 yr. Serum estradiol and progesterone were lower in +/- mice at all ages, and testosterone was several-fold higher in 7-mo-old and 1-yr-old +/- mice. Inhibin alpha (Western blot) appeared to be lower in +/- ovaries at all ages. FSH-R (FSH* binding) declined steadily from 3 mo and reaching the lowest point at 1 yr. LH receptor (LH* binding) was high in the 1-yr-old ovary, and expression was localized in the stroma and interstitial cells. Our findings demonstrate that haploinsufficiency of the FSH-R gene could cause premature exhaustion of the gonadal reserves previously noted in these mice. This is accompanied by age-related changes in the hypothalamic-pituitary axis. As these features in our FSH-R +/- mice resemble reproductive failure occurring in middle-age women, further studies in this model might provide useful insights into the mechanisms underlying ovarian aging.

Aging↗